Hematopoietic stem cell proliferation regulators and polynucleotides encoding the same

ABSTRACT

By analyzing hematopoietic stem cell proliferation regulators produced by stroma cells, a cDNA library is constructed and novel hematopoiesis-associated genes are isolated. Genes encoding the proteins as specified in the following (a) or (b): (a) having an amino acid sequence represented by one of SEQ ID NOS selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10 and 12; or (b) having an amino acid sequence derived from the amino acid sequence as defined in the above (a) by deletion, substitution or addition of one to several amino acids and having an activity of regulation hematopoietic stem cell proliferation.

FIELD OF THE INVENTION

The present invention relates to a novel hematopoietic stem cellproliferation regulators produced by a bone marrow stromal cell which isconsidered to form a hematopoietic microenvironment (niche) and regulatethe proliferation and the differentiation of hematopoietic stem cells orhematopoietic precursor cells via hematopoietic factors and adhesionmolecules, as well as a gene (polynucleotide) encoding the same.

BACKGROUND OF THE INVENTION

Hematopoietic stem cells present in a bone marrow, and mesencymal stemcells serve to maintain an adult body life by being differentiated overtheir entire life span into respective blood cells, bones, cartilages,fat cells and the like the supply of which is done by them continuously.Recently, these tissue-specific stem cells were reported to be capableof being differentiated also into the cells of multiple organs (nerve,liver, lung, intestinal tracts, cardiac muscles, muscles and the like)under a certain condition, and it is considered to be very important tounderstand their proliferation mechanisms also in view of theapplication to a transplantation therapy and a regenerative medicine.

A large number of attempts have been made heretofore to accomplish an invitro amplification of a hematopoietic stem cell, and some of them weresuccessful to some extent. In bone marrow stromal cells as considered toform a hematopoietic microenvironment (niche) and regulate proliferationand differentiation of hematopoietic precursor cells via hematopoieticfactors and an adhesion molecules. Those reported as the hematopoieticfactors produced by the stromal cell are cytokines such as IL-3 andIL-6, SCF, receptor-type tyrosine kinase ligands such as an Flt-3 ligand(Gibson, F. M. et al., Br J Haematol 1995), a differentiation-inhibitingfactor Notch ligand jagged-1 (Varnum-Finney, B. et al., Blood 1998).

However, most of the membrane-binding factors and adhesion moleculesrequired for the hematopoiesis described above are considered to beunidentified. While it is impossible to culture a hematopoietic stemcell for a prolonged period only by combining currently known factorswith each other, such a culture is possible for a certain time period inthe presence of stromal cells which are hematopoiesis-supporting cells,suggesting that the stromal cells may express hematopoiesis-relatedproteins which have not been identified.

SUMMARY OF THE INVENTION

Accordingly, an objective of the invention is to solve theabove-mentioned problems by analyzing hematopoietic cellproliferation-regulating factors produced by stromal cells, constructinga cDNA library and isolating novel hematopoiesis-related genes wherebyenabling a future application to an in vitro amplification of thehematopoietic stem cells, a transplantation therapy againsthematopoietic malignancies regenerative therapy care using stem cellsand a gene therapy.

We made an effort to accomplish the above-mentioned objective andfinally discovered, in a bone marrow stromal cells, novel genes encodingproteins having a hematopoietic stem cell proliferation-regulatingactivity, whereby establishing the invention.

Thus, the invention relates to a gene encoding a protein (a) or (b):

-   (a) an amino acid sequence represented by one SEQ ID No. selected    from the group consisting of SEQ ID Nos. 2, 4, 6, 8, 10 and 12,-   (b) a protein having a hematopoietic stem cell    proliferation-regulating activity which consists of an amino acid    sequence formed as a result of a deletion, substitution or addition    of one or several amino acids in the amino acid sequence (a).

The invention also relates to a gene comprising a DNA (a) or (b):

-   (a) a DNA consisting of the base sequence represented by one SEQ ID    No. selected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9    and 11,-   (b) a DNA encoding a protein having a hematopoietic stem cell    proliferation-regulating activity which hybridizes under a stringent    condition with a DNA consisting of the base sequence (a).

As used herein, the term “hematopoietic stem cellproliferation-regulating activity” includes an ability of amplifying orincreasing the proliferation of a hematopoietic stem cell and an abilityof rather inhibiting the proliferation of the hematopoietic stem cell,and means any function for exerting some effect on the proliferation ofthe hematopoietic stem cell. The term “hematopoietic stem cell” meansnot only a hematopoietic stem cell in its narrow sense but alsomyelocytic, erythroblastic, megakaryocytic and lymphocytic precursorcells which have been differentiated once and can be detected forexample in the Cobblestone area forming cell (CAFC) described below.

The invention also relates to a protein encoded by such a gene or DNA.Since the 6 proteins which have been identified for the first time bythe invention are derived from a stromal cell and each have ahematopoietic stem cell proliferation-regulating activity as shown inExamples described below, they are designated as SDHF (Stromal cellDerived Hematopoietic Factor)-1, SDHF-2, SDHF-3, SDHF-4, SDHF-5 andSDHF-6. The base sequences encoding these proteins and the correspondingamino acid sequences are represented by SEQ ID Nos. 1 and 2, SEQ ID Nos.3 and 4, SEQ ID Nos. 5 and 6, SEQ ID Nos. 7 and 8, SEQ ID Nos. 9 and 10and SEQ ID Nos. 11 and 12.

The invention further relates to a recombinant expression vehiclecomprising at least any one of the genes described above, a transformantobtained by the transformation with said expression vehicle, and amethod for producing any of the proteins described above comprising theculture of said transformant.

The invention further relates to a method for regulating thehematopoietic stem cell-proliferating activity of a stromal cellcomprising the transformation of said stromal cell with at least any oneof the genes described above. In said transformed stromal cell, a geneof the invention is expressed and its hematopoietic stem cellproliferation-regulating activity results in an amplification orincrease or reduction in the hematopoietic stem cell-proliferatingactivity of said stromal cell. The transformation can be conducted forexample by a method known to those skilled in the art using arecombinant expression vehicle described above.

The base sequence encoding the amino acid sequence represented by SEQ IDNo. 13 has already been reported (1999, Genomics 61 (1), pp37-43), andreferred to as an ISLR whose function has never been known heretofore,but it has proven by us for the first time here to have a hematopoieticstem cell proliferation-regulating activity as shown in the EXAMPLESdescribed below.

Accordingly, the invention relates to a method for modifying thehematopoietic stem cell proliferation-regulating activity of a stromalcell consisting of a transformation of said stromal cell with a geneencoding a protein (a) or (b)

-   (a) an amino acid sequence represented by SEQ ID No. 13,-   (b) a protein having a hematopoietic stem cell    proliferation-regulating activity which consists of an amino acid    sequence formed as a result of a deletion, substitution or addition    of one or several amino acids in the amino acid sequence (a). The    transformation can be conducted for example by a method known to    those skilled in the art using a recombinant expression vehicle    described above.

The invention further relates to a composition for regulating ahematopoietic stem cell proliferation comprising as an active ingredientat least one protein selected from SDHF-1 to SDHF-6 and ISLR describedabove.

As used herein the term “hematopoietic stem cell-proliferating activity”means a function for exerting some effect on the proliferation of ahematopoietic stem cell, and the term “regulation” means any change insaid activity such as an amplification or increase, or rather inhibitionand the like.

The invention further relates to various antibodies such as polyclonalantibodies and monoclonal antibodies directed to respective proteinsaccording to the invention such as SDHF-1, -2, -3, -4, -5 and -6described above.

FIG. 23 shows schematic views of the molecular structures of SDHF-1 toSDHF-6 according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of SDHF-1 (SEQ ID NO: 2).

FIG. 2 shows the amino acid sequence of SDHF-2 (SEQ ID NO: 4).

FIG. 3 shows the amino acid sequence of SDHF-3 (SEQ ID NO: 6).

FIG. 4 shows the amino acid sequence of SDHF-4 (SEQ ID NO: 8).

FIG. 5 shows the amino acid sequence of SDHF-5 (SEQ ID NO: 10).

FIG. 6 shows the amino acid sequence of SDHF-6 (SEQ ID NO: 12).

FIG. 7 shows the results obtained by a micro array method.

FIG. 8 shows the results obtained by a northern blotting.

FIGS. 9 a and 9 b show the results obtained by a western analysis.

FIG. 10 shows the results obtained by an immunostaining method.

FIG. 11 shows the results of a CAFC analysis.

FIG. 12 shows the results of the post-translational modification of theproteins of genes SDHF-1 and SDHF-5 of the invention detected by thewestern analysis. The symbol “★” indicates a mature protein whosemolecular weight became larger as a result of the addition of a sugarchain, the symbol “▴” indicates a protein on the way of the productionbefore the addition of the sugar chain, the symbol “▾” indicates aresidual protein after a cleavage, and the symbol “●” indicates asecretory protein.

FIG. 13 shows the results of the detection by the methionine labeling ofa secretory protein in a cell culture supernatant. The symbol “●”indicates a secretory protein, and “→” indicates a protein secreted inthe culture supernatant after a cleavage.

FIGS. 14 a, 14 b , and 14 c show photographs of the electrophoresisshowing the results of the assay of the expression site of a gene of theinvention by an RT-PCR method.

FIG. 15 shows the results of an assay of the stem cell-supportingfunction of a gene of the invention by an LTC-IC method.

FIG. 16 shows a photograph of the electrophoresis of an SDHF-4extracellular region/recombinant protein as being purified by a proteinA column.

FIG. 17 is a graph showing that the SDHF-4 extracellularregion/recombinant protein has an ability of maintaining thenon-differentiated condition of a mouse hematopoietic stem cell.

FIG. 18 shows a microscopic photograph (magnification: ×40) showing thatSDHF-6, when expressed highly in an OP9 cell using a retrovirusexpression vector pMX-puro, has an ability of inducing thedifferentiation into a fat cell efficiently after about four-weekculture.

FIG. 19 shows a photograph of a western analysis showing the results ofthe experiment in which an antibody directed to a partial peptide ofSDHF-4 was employed to allow SDHF-4 to be expressed in a CHO-k1 cellusing a pSSRα-bsr vector.

FIGS. 20 a and 20 b show the results of a FACS calibur (BectonDickinson) analysis of a CHO-k1 cell allowed to express SDHF-4 afterlabelling the purified antibody described above with FITC.

FIGS. 21 a and 21 b show the results of a sorting of a cell expressingSDHF-4 using a FACS Vantage (Becton Dickinson) after staining amyelocyte of a C57BL/6J mouse using the same antibody.

FIG. 22 shows a microscopic photograph of the above-mentioned cellselected by the sorting (magnification: ×200).

FIG. 23 shows a schematic view of the molecular structures of SDHF-1 toSDHF-6.

BEST MODE FOR CARRYING OUT THE INVENTION

A gene or DNA of the invention can be prepared for example by a methodshown in the EXAMPLES described below based on a cDNA library preparedfrom an mRNA derived from a stromal cell such as a mouse stromal cellline OP9 which has been stimulated by a leukemia-inhibiting factor (LIF)during culture.

Alternatively, it can be prepared also by a chemical synthesis using aprocedure known in the art based on the sequence information disclosedin this specification. Those skilled in the art may accomplish thedeletion, substitution or addition of one or several amino acids in acertain amino acid sequence by means of a method known per se in theart.

In the invention, a specific gene or DNA can be hybridized under astringent condition with regard to various parameters such as saltconcentrations at a suitable temperature in a buffer solution known tothose skilled in the art.

An example of the DNA which hybridizes under such a stringent conditionwith a gene or DNA of the invention and which encodes a protein having ahematopoietic stem cell proliferation-regulating activity is a DNA whosehomology with such a gene is 70% or more, preferably 90% or more.

A DNA obtained by binding a gene or DNA of the invention appropriatelyto any of various sequences known to those skilled in the art in a generecombination procedure such as regulatory factors including promotersand enhancers, restricted enzyme sites, selection marker (marker enzyme)gene and the like is also encompassed by the invention.

An expression vehicle such as a recombinant plasmid vector having agene, DNA or a DNA molecule of the invention described above or arecombinant virus vector employing a retrovirus and the like, and atransformant of a microorganism such as Escherichia coli and aneukaryotic cell such as a mouse COS-7 cell or a mouse stromal cell OP9,which contains such an expression vehicle and has thus been transformed,are also encompassed by the invention. These DNA molecules, expressionvehicles and transformants can readily be prepared by a method known inthe art. In addition, a protein of the invention can be readily producedby those skilled in the art by culturing an above-mentioned transformantby a known method and purifying the culture product.

A hematopoietic stem cell proliferation regulation activatingcomposition of the invention comprising a protein described above as anactive ingredient may also contain, as appropriate, other cytokinesknown as hematopoietic factors, as well as suitable carriers andauxiliary agents known in the art. The amounts and the ratios of theprotein of the invention as an active ingredient and other componentsmay be selected by those skilled in the art depending on the purpose ofuse.

The antibodies such as polyclonal and monoclonal antibodies of theinvention directed to the respective proteins of the invention such asSDHF-1, -2, -3, -4, -5 and -6 can be produced by any known method. Forexample, a polyclonal antibody can readily be produced by inoculatingeach protein described above or its partial peptide as an immunogen toany experimental animal such as a rabbit. A monoclonal antibody canreadily be obtained by subjecting an antibody-producing cell preparedsimilarly using such an immunogen to a cell fusion with a suitableparent cell (immortalized cell line) by a method known in the art toyield a hybridoma followed by screening the hybridoma for a clone whichreacts specifically with each protein. The partial peptide correspondsto any partial amino acid sequence in the respective protein, and thenumber of the amino acids is not limited particularly but preferably isat least 10, and usually is within the range from 10 to 20. In view ofthe reactivity with each protein, it is preferable to select a partialpeptide employed as an immunogen from the extracellular region of therespective protein.

Such an antibody can be used in a procedure for isolating or purifyingvarious adult cells such as bone marrow stem cells, mesencymal stemcells and nervous stem cell as well as stem cell-supporting cells(stromal cells) such as a bone marrow hematopoiesis-supporting cells.Such an isolation or purification may be conducted by various meansknown to those skilled in the art, such as an affinity columnpurification or a FACS sorting.

EXAMPLES

The invention is further described referring to the following EXAMPLES,which are not intended to restrict the technical scope of the invention.

cDNA Library Construction and Signal Sequence Trap (SST) Method

A mouse stromal cell line OP9 is incubated in Alpha-MEM mediumsupplemented with 20% fetal bovine serum. APLAT-E cell (2000, Morita etal., Gene Therapy vol. 7, 1063-6) was incubated in DMEM supplementedwith 10% fetal bovine serum. The extraction of polyA RNA from the OP9cell was conducted using an Invitrogen FAST TRACK 2.0 mRNA Isolation Kitin accordance with the protocol attached thereto. As a starting materialfor a cDNA library, 5 μg of polyA RNA extracted from 1×10⁸ OP9 cellsstimulated for 60 minutes at 37° C. with 10 ng/ml of mouse leukemiainhibitory factor (LIF) was employed. The cDNA library was constructedusing a SUPERSCRIPT II (Invitrogen) using random hexamers as primers.The resultant cDNA library was combined with a BstXI adapter primer(Invitrogen) and integrated into the BstXI site of a retrovirus vectorpMX-SST vector (1999, Kojima et al., Nat Biotech vol. 17, p. 487) toconstruct a plasmid library.

The cDNA library obtained was introduced into a PLAT-E cell using FuGene6 (Roche), and then recovered in the form of a retrovirus in thesupernatant. The resultant retrovirus cDNA library was infected to aBa/F3 cell which is an interleukin 3 (IL-3)-dependent mouse pro-B cell.The pMX-SST vector has a signal peptide-deficient constantly activatedc-Mp1 gene, and the cmp1 gene is activated when a library-derived genehas a signal peptide and consequently the Ba/F3 cell is immortalized inthe absence of IL-3 (1999, Kojima et al., Nat Biotech vol. 17, p.487-90). The retrovirus library-infected Ba/F3 cell was deprived ofIL-3, and incubated in a 96-well plate at the density of 1000 cell/well,and then the proliferated cells were isolated and the genome DNA wasextracted. Since a signal peptide-carrying gene has been inserted intothe genome DNA of the Ba/F3 cell via the retrovirus, it was recovered bya genome DNA PCR (LA-Taq, TAKARA, 98° C. for 20 sec. 68° C. for 300sec.: 35 cycles) using the primers (GGGGGTGGACCATCCTCTA,CGCGCAGCTGTAAACGGTAG) designed based on the sequence of the pMX-SSTvector.

Gene Sequencing

Since a signal peptide-carrying clone was obtained as a DNA fragment bythe PCR described above, its DNA sequence was determined using an ABI373A sequencer. The resultant PCR fragment was purified by a PCRpurification kit (Quiagen) to be deplete the primers, and the sequenceswere determined by a dye termination method in accordance with theprotocol by ABI. The resultant sequence information was analyzed by anNCBI BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) whereby judgingwhether the sequences were known or unknown. A part of an unknownsequence was determined by a 3′ RACE method using primers designed basedon the sequences obtained. The 3′ RACE method is a strategy foridentifying an unknown gene to the extent of the polyA region by a PCRmethod using the primers designed on the basis of the above-mentionedPCR clone sequences as well as oligo dT primers. The DNA sequence of anyresultant PCR fragment was determined as described above using an ABI373A sequencer.

As a result, the genes of 234 clones in total including known andunknown ones were isolated by the SST method described above. Theyincluded 181 known genes, 42 unknown genes and 11 genes which could notbe analyzed. The known genes included 49 proliferation factors, 23receptors, 71 adhesion molecules and 38 others.

Among the unknown genes, 6 SDHF (stromal cell derived hematopoieticfactor) genes of the invention and ISLR were included. As shown in FIGS.1 to 6 and in SEQ ID No. 13, the N terminal of the amino acid sequenceencoded by 6 SDHF and ISLR genes contained a signal peptide (regionsurrounded by a rectangular frame). The results obtained as describedabove are summarized in Table 1 and Table 2.

TABLE 1 SDF-1-alpha 27 ISLR (immunoglobulin superfamily containingleucine-rich repeat) 11 ADAMTS-1 (secretory protein, metalloprotease) 10SDHF-1 7 collagen a1(V) 7 Fractalkine 7 amyloid beta protein precursor 6collagen alpha1 (VI) 6 gp130 6 thrombospondin 1 6 CCK4 (RTK), homologue5 collagen, alpha-2 collagen VI 5 collagen, pro-alpha1 (II) collagenchain 5 fibronectin 5 SPARC-related protein (SRG) 5 syndecan 5 amyloidprecursor-like protein 2 4 BiP, immunoglobulin heavy chain bindingprotein 4 insulin-like growth factor binding protein 4 4 interleukin 1receptor accessory protein. 4 protein disulfide isomerase (ERp59) 4Cyr61, CTGF, IGFBP10 3 lysyl oxidase (Lox) 3 SDHF-2 2 collagen, alpha-2type IV collagen 2 collagen, pro-alpha-2(I) collagen 2 collagenase, typeIV collagenase 2 osteopontin 2 P2B/LAMP-1 2 PDGFR beta 2 vimentin 2SDHF-3 1 SDHF-4 1 SDHF-5 1 SDHF-6 1 SDHF-7 1 SDHF-8 1

TABLE 2 Unknown clones ISLR 11 SDHF-1 7 SDHF-2 2 SDHF-3 1 SDHF-4 1SDHF-5 1 SDHF-6 1 SDHF-7 1 SDHF-8 1 others 16 42 Known clones FACTORSSDF-1-alpha 27 Fractalkine 7 insulin-like growth factor binding protein4 4 Cyr61, CTGF, IGFBP 10 3 lysyl oxidase (Lox) 3 osteopontin 2 alphainhibin 1 S1-5, T16 homologue, 1 STRA-1/EFLN B2 1 49 RECEPTORS gp130 6CCK4 (RTK), homologue 5 IL-1 receptor accessory protein. 4 PDGFR beta 2Fe receptor 2 clone: 2-63 1 FGF receptor 1 LDL receptor 1 ROBO-1 1 23Adhesion & Matrix proteins ADAMTS-1 (secretory protein, metalloprotease)10 collagen a1(V) 7 amyloid beta protein precursor 6 collagen alpha1(VI) 6 thrombospondin 1 6 collagen, alpha-2 collagen VI 5 collagen,pro-alpha1 (II) collagen chain 5 fibronectin 5 SPARC-related protein(SRG) 5 collagen, alpha-2 type IV collagen 2 collagen, pro-alpha-2(I)collagen 2 collagenase, type IV collagenase 2 cadherin-11 (OSF-4) 1calpain small subunit 1 calumenin 1 collagen, type 1 procollagenC-proteinase enhancer protein 1 entactin/nidogen 1 extracellular matrixassociated protein (Sc1) 1 metalloproteinase 1 1 nucleobindin 1thrombomodulin 1 type 1 procollagen C-proteinase enhancer protein 1 71Others 31 not membranous 7 TOTAL 223Microarray Method

All PCR fragments obtained by the signal sequence trap method werespotted onto glass slides (procedure heretofore being conducted byHOKKAIDO SYSTEM SCIENCE), and the change in the expression wasinvestigated using a fluorescence-labeled cDNA probe prepared from thecells adjusted under the condition before and after the stimulation ofthe OP9 cells with 10 ng/ml LIF at 37° C. for 60 minutes. The probe wassubjected to a reverse transcription using a SuperScript II employingoligo dT primers from the polyA RNA extracted as described above, andthen purified using a Microcon 30 (Millipore) labeled by means of theintegration of a Fluorolink Cy-dUTP. The results obtained are shown inFIG. 7.

Northern Blotting

The OP9 cells were stimulated with 10 ng/ml of LIF at 37° C. for 60minutes, and then after 0 minutes, 30 minutes, 1 hour, 3 hours, 8 hoursand 24 hours, the total RNA was extracted using a Trizol reagent(Invitrogen) in accordance with the protocol. The total RNA thusextracted was examined for the concentration, and then a 10 μg aliquotwas subjected to an electrophoresis on a 1% agarose gel, transferredonto a Hybond N (Amersham), and subjected to a northern blotting by astandard method using the respective gene DNA fragment as a probe. As aresult, an increase in the expression of each of SDHF-1, SDHF-2 andSDHF-5 genes was observed as evident from FIG. 8. On the contrary,SDHF-6 gene exhibited a reduction in the expression.

Western Blotting

From the sequence of the entire length of a gene thus obtained, an aminoacid-encoding region (open reading frame: ORF) was determined, and itscarboxyl terminal was tagged with a FLAG tag sequence (DYKDDDDK) by aPCR method. All of these cDNAs were subcloned into an expression vectorpUC-CAGGS which was then expressed transiently in COS-7 cells usingFuGene 6 (Roche). The western blotting was conducted as reportedpreviously (1995, Ueno, JBC Vol. 270, pp. 20135-42). The cells weresolubilized with a Triton lysis buffer (0.5% (v/v), Triton X-100, 50 mMTris-HCl, pH 7.4, 2 mM PMSF, 10 U/ml aprotinin, 1 mM EDTA) and then a 50μg lysate was subjected to an SDS-polyacrylamide gel electrophoresis(PAGE), transferred to an Immobilon-P membrane, and then detected usingan anti-FLAG antibody, M2 (Sigma). As a result, any of the genes of theinvention was expressed in the COS7 cells and exhibited the bands in thewestern blotting.

Immunostaining

Genes tagged with a FLAG peptide were expressed transiently in the COS-7cells using a FuGene reagent (see above), fixed in a 3.7% formaldehyde,subjected to a permeabilization with a 1% NP40, and stained with ananti-FLAG antibody (secondary antibody: Texas Red-labeled anti-mouseantibody, Jackson). The nucleus was stained with DAPI (FUNAKOSHI). Theresults are shown in FIG. 10.

Cobblestone Area Forming Cell Assay: CAFC Assay

A gene obtained as described above in the OP9 cells was transduced intoa retrovirus vector (pMX-puro), which were inoculated to a 6-well dishat the density of 2.5×10⁵, and incubated overnight and then inoculatedwith 1×10⁵ of the bone marrow cells obtained from the femoral bones of 5to 8-week old C57BL/6mice, and the number of the CAFCs formed wascounted after 6 days.

Results of CAFC Analysis

Hematopoietic cells proliferating underneath a stromal cell layer uponco-culturing the stromal cells and the bone marrow cells are referred toas a cobblestone area forming cell (CAFC). An increased number of theCAFCs formed is largely means that the stromal cell amplifies theimmature hematopoietic cells. Nevertheless, an increased CAFC does notmean an amplified hematopoietic stem cell in an exact sense, since thecells consisting the CAFC include not only the hematopoietic stem cellbut also the cells which have been differentiated once, such asmyelocytic, erythroblastic, megakaryocytic and lymphocytic precursorcells. The gene described above was introduced into the OP9 cells of amouse myelic stromal cell line having a hematopoiesis-supporting abilityusing a retrovirus vector pMX-puro, and the cells imparted with a drugresistance by 5 μg/ml puromycin was examined for their CAFC-formingability. As a result, ISLR exhibited the highest level, and slightlyhigher levels were also exhibited by SDHF-1, SDHF-2, SDHF-4 and SDHF-5,as shown in FIG. 11. These findings suggest the hematopoietic precursorcell-amplifying ability of these genes. On the contrary, SDHF-3exhibited the inhibition of the CAFC-constituting ability, and wasconsidered to have a hematopoietic stem cell proliferation-inhibitingability.

Detection of Secretory Protein in Cell Culture Supernatant by MethionineLabeling

1×10⁵ COS-7 cells were inoculated into a 6 cm dish, which was thenincubated overnight. The genes were introduced by a FuGene method(Roche). The carboxyl terminal of any of these genes is tagged with aFLAG tag, and a protein expressed can be detected by a western analysisusing an anti-FLAG antibody as described above. After incubation for 36hours, the supernatant was removed, and the cells were washed twice withPBS, combined with 0.5 ml of DMEM (Methionine, Sigma) supplemented with0.5 mCi [³⁵S] methionine (ICN, TRAN-S), and incubated for 4 hours. Thecell components were removed from the culture supernatant bycentrifugation, and the proteins were concentrated using a Micron 10(Millipore). After running in a 15% SDS-PAGE, the labeled proteins weredetected using a BAS2000 (FUJI FILM).

As a result, SDHF-1 and SDHF-5 exhibited the protein secretion to theculture supernatant as shown in FIG. 12 and FIG. 13. Since these genesare membrane proteins structurally and the secretory proteins aresmaller by about 20 kDa than the mature protein detected by the westernanalysis, these secretory proteins were considered to have undergone thecleavage of the transmembrane region of the carboxyl terminals. Since anSCF is known to be a proliferation factor of the type which is secretedas a result of the cleavage of the transmembrane region of the carboxylterminal and such findings were not shown by an adhesion protein, theproteins encoded by the genes of the invention are suggested strongly tobe the proteins of the type which serve as humoral factors to transmitthe signals to other cells.

Assay of Expression Site by RT-PCR

Based on the DNA sequences of respective SDHF genes, the primers weredesigned and a PCR (LA-Tag, TAKARA) was conducted using Multiple TissuecDNA (MTC) panels (Clontech) as templates. The cells in a bone marrowwere sorted by a FACS Vantage (Becton Dickinson) usingfluorescence-labeled monoclonal antibodies (anti-B220, anti-CD3,anti-Gr1, anti-MAC1, anti-Sca1 antibodies, Phamingen), subjected to anRNA extraction with a Trizol reagent (Invitrogen) followed by an RTreaction using random hexamers, and then subjected to the PCR similarly.

As a result, all SDHF genes exhibited the expression in the OP9 cells asshown in FIG. 14. SDHF-4 exhibited the expression localized especiallyin the brain and the bone marrow, in which the expression was localizedin the stromal cells interestingly.

Assay of Stem Cell-Supporting Ability by Long Term Culture-InitiatingCells Assay (LTC-IC Method)

Since the CAFC method described above only reflects thehematopoiesis-supporting function over a relatively short period, anLTC-IC method was conducted for the evaluation of the stemcell-supporting function over a prolonged period. A gene isolated asdescribed above was expressed highly in the OP9 cells using a retrovirusexpression vector pMX-puro whereby conducting an LTC-IC method.

First, a hematopoietic stem cell was purified. Thus, the bone marrowcells were obtained from the femoral bones of 6 to 8-week old C57BL/6mice, and the mononuclear cell were separated by centrifugation by aFicoll method (specific gravity: 1.100). After washing with PBS, thecells were reacted with a biotin-labeled primary antibody (anti-Lineageantibody cocktail: anti-CD3, anti-CD4, anti-CD8, anti-B220, anti-Ter119,anti-Gr1, anti-Mac1 antibodies, all from Phamingen) followed bystreptoavidin-labeled magnetic beads each at 4° C. for 15 minutes. AMACS method was employed to recover the column-passing fraction (LIN(−)cells), which was washed by centrifugation, reacted further with aPE-labeled anti-Sca1 antibody, FITC-labeled anti-c-kit antibody,Per-CP-Cy5.5-labeled streptoavidin (all from Phamingen) at 4° C. for 15minutes, subjected to a FACS Vantage (Becton Dickinson) to obtain LIN(−), Sca1 (+) and c-kit (+) fractions (the cells thus obtained weredefined as KSL cells). cDNAs obtained by adding FLAG peptides to thecarboxyl terminals of SDHF-1 to 6 were transduced to the OP9 cells usinga retrovirus vector (pMX-puro) to establish stably expressing celllines, 2.5×10⁵ of which were inoculated to a 6-well dish, which wereincubated overnight, inoculated with 100 KSL cells, which were thenincubated for 3 weeks. Thereafter, the cells were recovered andinoculated, after 2-fold dilution in 8-well pairs, to a 96-well dish,each well of which had been inoculated with 3000 OP9 cells which hadbeen irradiated with γ-ray at 20 Gy. After 5 weeks, the cells wererecovered from each well, inoculated to a methyl cellulose medium (IMDM,1% methyl cellulose, 15% fetal bovine serum, 1% bovine serum albumin, 3U/ml human erythropoietin, 10 ng/ml human IL-6, 10 ng/ml mouse IL-3, 100ng/ml mouse SCF, 10 μg/ml bovine insulin), and then evaluated after 12days on the basis of the presence or absence of the blood cell colonies(CFU-C). The frequency of the stem cells were analyzed by an L-Calcsoftware (Stemcell).

As a result, a marked hematopoiesis-supporting function was exhibited bythe SDHF-4 gene as evident from FIG. 15. The hematopoiesis-supportingfunction of this gene was revealed.

Mouse Hematopoietic Stem Cell-Amplifying Effect of SDHF-4 ExtracellularRegion/Recombinant Protein

The extracellular transmembrane region of SDHF-4 was assumed (aminoacids 524 to 540) using a PSORT II program(http://psort.ims.u-tokyo.ac.jp/form2.html), and a human immunogloblinFc region and a fusion protein (SDHF-4-Δ TM-Fc) were formed in theextracellular region (amino acids 1 to 523), and subcloned into anexpression vector pSSRα, which were stably introduced into CHO-k1 cells.A culture in a serum-free medium CD-CHO (Invitrogen) followed by thepurification of the supernatant on a Protein A column yielded fusionrecombinant proteins (FIG. 16). Mouse stem cells were cultured similarlyto the LTC-IC method as the mouse bone marrow LIN (−), Sca1 (+) andc-kit (+) fractions (KSL cells), each 100 cells of which were incubatedin a Stem Span medium (StemCell) supplemented with SCF (50 ng/ml), IL-3(10 ng/ml), IL-6 (10 ng/ml) and SDHF-4-Δ TM-Fc (100 ng/ml). After 7days, the cells were recovered, and examined for the expression of LIN,Sca1 and c-kit using a FACS Calibur.

As a result, the LSK cells incubated in the StemSpan medium supplementedwith SCF, IL-3 and IL-6 exhibited not only a LIN (−) cell ratio whichwas higher in the presence rather than the absence of 100 ng/ml ofSDHF-4-Δ TM-Fc but also increased ratios of LIN (−), Sca1 (+) and c-kit(+) fractions. These findings suggest that the SDHF-4 extracellularregion/recombinant protein has an ability of maintaining thenon-differentiated state of the mouse hematopoietic stem cells.

Fat Cell Differentiation Assay and Oil Red O Staining

Each of the cell into which an SDHF-6 gene had been transduced via aretrovirus vector (pMX-puro) and the cell into which only the vector hadbeen transduced was incubated continuously in a confluent state withoutany subculture over a period of 4 weeks or longer in the presence ofAlpha-MEM+20% fetal bovine serum. The cells were fixed with 10% formalinand then stained with an Oil Red 0.

As a result, the OP9 cells over expressing SDHF-6 by using theretroviral vector pMX-puro were revealed to differentiate into fat cellswith a high efficiency after the incubation for about 4 weeks. This genewas considered to be involved in the differentiation into the fat cellsvia the effect on the mesencymal cells rather than on the hematopoieticstem cells.

Preparation of Polyclonal Antibody Directed to SDHF-4 and Isolation ofmyelic Hematopoiesis-Supporting Cell Therewith

Based on the amino acid sequence of SDHF-4, a synthetic peptide(GYMAKDKFRRMNEGQVY (SEQ ID NO: 14) (corresponding to the amino acids 32to 48)) was designed, and immunized to a rabbit to prepare a polyclonalantibody.

This peptide was conjugatal with an epoxy-activated agarose gelSEPHAROSE 6B) (Pharmacia) to prepare an antigen column, which was usedto purificate a polyclonal antibody, as an affinity chromatography. Awestern analysis using this antibody while comparing the cell expressingSDHF-4 obtained by transducing a CHO-k1 cell with a pSSRα-bsr vector(SDHF4-c112) with the cell into which only the vector had beenintroduced (mock) revealed that an SDHF-4 gene product could berecognized in a very specific manner (FIG. 19). 1 mg of the resultantpurified antibody was labeled with FITC using a LinKit Fluoro-Link (ISL)and the same cell was analyzed by a FACS Calibur (Becton Dickinson), andthe results indicated that this antibody can be utilized also in a flowcytometry (FIG. 20: O-line: mock, ▪-line: SDHF4-c112). Using thisantibody, the bone marrow cells of a C57BL/6J mouse were stained, andsubjected to a FAGS Vantage (Becton Dickinson) whereby sorting andrecovering the cells expressing SDHF-4 (FIG. 21), which deposited onto aculture dish and were revealed morphologically to be mesencymal cellscorresponding to bone marrow hematopoiesis-supporting cells (FIG. 22).

INDUSTRIAL APPLICABILITY

According to the invention, a stromal cell-derived novelhematopoiesis-related gene can be isolated successfully, and wasrevealed to have a hematopoietic stem cell proliferation-regulatingactivity. As a result, a wide range of the application, including an invitro amplification of hematopoietic stem cells, a transplantationtherapy against a malignant tumor of a hematopoietic tissue, aregenerative medicine using the stem cell, a gene therapy,immunotherapy, cell transplantation, treatments of a neuropathy(Alzheimer's disease, cerebral infarction, degenerative neuropathy andthe like), hepatic disease (cirrhosis), pulmonary disease, myocardialdisease, diabetes, bone disease, chronic renal failure and the like,became possible.

1. A gene encoding a protein comprising an amino acid sequencerepresented by SEQ ID No.
 8. 2. A gene comprising a DNA consisting ofthe base sequence represented by SEQ ID No.7.
 3. A protein encoded by agene according to claim 1 or
 2. 4. A recombinant expression vehiclecomprising at least one gene according to claim 1 or
 2. 5. A recombinantexpression vehicle according to claim 4 which is a recombinant plasmidvector.
 6. A recombinant expression vehicle according to claim 4 whichis a recombinant retrovirus vector.
 7. A transformant obtained by atransformation by an expression vehicle according to claim
 4. 8. Atransformant according to claim 7 which is a COS-7 cell.
 9. Atransformant according to claim 7 which is a stromal cell.
 10. A methodfor producing a protein comprising culturing a transformant according toclaim 7.